The invention is in the field of tissue engineering and culture systems and is directed primarily towards reconstruction of in vitro cell-based models for use as animal alternatives in irritancy, toxicity and drug efficacy testing. In particular, the invention relates to an artificial cornea.
Animal tests are used for screening new chemicals, drugs, finished products or their ingredients for potential ocular irritancy. The historic test that is still in widespread use is the Draize rabbit eye irritancy test. The Draize test was developed in 1944 and involves the introduction of 0.1 ml of a test substance into the lower conjunctival sac of albino rabbit eyes (Draize et al. 1944). Responses of the cornea, conjunctiva and iris are graded using a numerical scoring system. The cornea is scored for opacity that is induced by irritants. The conjuctivum is scored for an angiogenic response (such as redness caused by blood vessel dilation) while the iris gives a neurological response (i.e., contraction or dilation). Of these, an indication of corneal clarity/opacity is the most relied upon indicator for irritancy. Although still regarded as the definitive test available, the Draize test suffers not only from strong resistance from the public, but also because the rabbit eye responds differently to various traumas than does the human eye.
Animal testing is also used in the development of ophthalmic drugs. Again, differential responses between animal models, such as rabbits and humans, are well documented for drugs ranging from analgesics for pain and antibiotics, to steroid and non-steroid preparations that modify wound healing responses.
Enucleated whole eyeballs from rabbits and cattle have been tested as possible Draize alternatives. Among them are the Bovine Eye Assay which measures corneal opacity/permeability from Merck Sharp and Dome laboratories. While enucleated rabbit and bovine eyes have shown a capacity to handle and identify severely irritating substances over a wide range of physical forms and solubilities, they are less sensitive for resolving responses to very mild and moderately irritating substances (Burton et al. 1981; Gautheron et al. 1992). In addition, these are animal derived tissues and therefore have the same accuracy problems and difficulty with extrapolation to humans as the Draize test. Moreover, the very short term viability of these corneas (tests are done on the day of enucleation) will not allow for longer term screening of more chronic effects of the milder test substances.
Organ cultured human corneas can be used for testing. Richard et al. (1991), Anderson et al. (1993) and Collin et al. (1995) have successfully organ cultured freshly obtained whole human corneas, allowing for investigations to be carried out on whole corneas. However, organ cultured corneas have a finite lifespan. The longest time recorded is 21 days (Anderson et al. 1993). More importantly, the availability of donors is sporadic and donor corneas are too heterogeneous to be useful as a practical model for drug testing.
Other alternatives that have been developed include the EYTEX(trademark) test from In Vitro International (formerly Ropak Laboratories) which is based on the quantification of opacity produced in a synthetic protein matrix on exposure to chemical irritants (Gordon, 1992); the Keratinocyte Neutral Red Uptake Bioassay from Clonetics Corporation based on the methodology of Borenfreund and Puerner (1985), the MTT assay using living skin equivalent from Advanced Tissue Sciences (Osborne et al. 1995), and the Chorioallantoic membrane vascular assay (Bagley et al., 1992). Trans-epithelial permeability assay can also be used as an in vitro assay for predicting ocular irritancy using MDCK cells (Martin and Stott, 1992). Others use an agar diffusion cytolysis as an alternative screen for the prediction of a corrosive ocular response using rabbit cornea fibroblasts (Galer, 1992).
Results indicate that while it may not be possible to use a single test to classify compounds as to irritation potential, some of the alternative test models may be useful in prioritizing and reducing the number of in vivo irritation tests conducted. However, all of the tests are dependent upon continued rabbit eye testing as definitive tests and also to develop validation databases to aid interpretation of the in vitro data.
Researchers have been attempting to reconstruct corneas in vitro from cell lines. Individual human corneal epithelial layers have been successfully maintained in culture as a stratified epithelium by Kahn et al. (1993) and Araki-Sasaki et al. (1995). These investigators had used corneal epithelial cell lines that were immortalized using a hybrid SV40-adenovirus. Successful reconstructions of corneas comprising the three main layers have also been recently reported by Minami et al. (1993) and Zieske et al. (1994). These corneas were reconstructed from either passaged primary cells from bovine (Minami et al. 1993), or mixed cultures of primary rabbit epithelial and stromal cells and an immortalized mouse endothelial cell line (Zieske et al. 1994).
To date, there have been no reports of successfully reconstructed human corneas based fully on human cell lines while mimicking the physiology of the human cornea and surrounding tissue.
Media used in preparing artificial corneas, or in culturing whole corneas, must be carefully selected to avoid detrimental effects to the desired process. Certain serum proteins will affect wound healing and hence a serum-free medium is desirable to allow optimal control of experimental conditions. Disclosed herein is a serum-free medium that may be used to prepare the cornea of the present invention, as well as for culturing whole corneas.
The present invention provides an artificial mammalian cornea which comprises:
a) an endothelium comprising primary or immortalized mammalian endothelium cells;
b) a stromal matrix;
c) an epithelium comprising primary or immortalized mammalian epithelium cells; and
d) at least one layer selected from Bowman""s membrane and Descemet""s membrane.
The present invention also provides a method for preparing an artificial mammalian cornea which comprises:
a) growing an endothelium which comprises primary or immortalized mammalian endothelium cells;
b) optionally layering a Descemet""s membrane on the endothelium;
c) forming a stromal matrix on the endothelium or on the Descemet""s membrane, if present;
d) optionally layering a Bowman""s membrane on the stromal matrix; and
e) growing an epithelium, which comprises primary or immortalized mammalian epithelium cells, on the stromal matrix or on the Bowman""s membrane, if present;
wherein at least one of Bowman""s membrane and Descemet""s membrane is present.
The present invention further provides a method for preparing an artificial mammalian cornea which comprises:
a) growing an epithelium which comprises primary or immortalized mammalian epithelium cells;
b) optionally layering a Bowman""s membrane on the epithelium;
c) forming a stromal matrix on the epithelium or on the Bowman""s membrane, if present;
d) optionally layering a Descemet""s membrane on the stromal matrix; and
e) growing an endothelium, which comprises primary or immortalized mammalian endothelium cells, on the stromal matrix or on the Descemet""s membrane, if present;
wherein at least one of Bowman""s membrane and Descemet""s membrane is present.
The present invention also provides a commercial package which comprises:
a) the cornea as described herein, and
b) instructions for its use for testing a substance for ocular irritancy, toxicity or drug efficacy, or transplantation.
The present invention also provides a serum-free culture medium which comprises at least one of each of a protease inhibitor, a growth factor, a substance which mediates the growth factor and a free-radical scavenger, and preferably a substance which prevents cellular contraction, as well as instructions for use of the medium in culturing a cornea, preferably a human cornea, in vitro.
The present invention also provides an artificial sclera which comprises primary or immortalized mammalian angiogenic cells.
Gene transfer and tissue culture techniques have been used to construct a cornea from immortalized human cell lines that is analogous to the human cornea in vivo. The corneal equivalent is preferentially surrounded by a sclera capable of mimicking inflammation responses.
Various different gene constructs have been introduced into the immortalized cell lines by transfection and/or retroviral infection to either overexpress, or block expression (antisense constructs) of, gene products in order to mimic different pathological conditions that occur in the cornea. This allows the development of in vitro corneal disease models for studying drug treatment efficacy.
The model can also be modified to render it suitable for use in transplantation. Also described is a medium that can be used to start and maintain cultures of primary cells and cell lines from cornea and other tissues. The medium is also suitable for the culture of corneas, both artificial and natural, in the absence of serum.
The artificial cornea is preferably surrounded by a matrix in which angiogenesis (formation of capillary-like structures) can occur in vitro. The artificial cornea is physiologically and functionally similar to the human cornea and has the potential to respond to ocular irritants, drugs and injuries. The surrounding angiogenic material plays the role of pseudo-sclera and thereby provides the ability to assess in vitro the angiogenic reaction to any chemical irritants, drugs or any injuries. The invention extends to a method for making the artificial cornea and a method for using the artificial cornea. Preferably, the corneal or scleral cells used, whether primary or immortalized, are of human origin.
The invention also provides a serum-free medium that has been used to successfully start and maintain the cell lines, as well as the artificial corneas. The medium may also be used in reconstruction of other body tissues that could be used as complementary in vitro models for alternative models to animal testing.
The artificial cornea can also be modified for transplantation by modification of the matrix compounds by physical and chemical treatments, and incorporation of matrix proteins or essential amino acid sequences and/or growth factors. Primary cells can be used until the safety of cells containing viral constructs for transplantation has been determined. In addition, cells from one or more layers may be omitted to allow for the host/patient""s cells to repopulate the transplanted matrix material.